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This article reports on a project investigating the impact of a new framework for Readers Theatre on teachers' professional skills. Twenty-one Islamic secondary English school teachers engaged in collaborative professional development training to experience and give feedback on the framework. The article describes the participants' affective and critical responses and perspectives as well as opportunities and challenges related to the new framework in infusing peace values into EFL (English as a Foreign Language) materials. This study provides fresh insight into the potential of Readers Theatre for teaching peace and conflict prevention in the Indonesian Islamic EFL secondary school context. [ABSTRACT FROM AUTHOR]

Although previous work explained internal and external cultural challenges impacting academic integrity in the Muslim world, to date, no study has specifically examined the attempts and practices by the universities to address these challenges. The objective of this paper was to understand the actions taken by academics and institutions in the Muslim world to address, prevent breaches of academic integrity, and to recommend improvement of these practices. To capture institutional efforts and practices, relevant literature from 2010 to 2021 was reviewed to gather evidence of practices of academic integrity in higher education in the Muslim world. The findings suggest a framework that can be used for evaluation of current practices of academic integrity in the Muslim world, to go beyond plagiarism-focussed prevention, detection, and punishments. [ABSTRACT FROM AUTHOR]

We learned a lot about both Carol and Sebla from this piece, and hope that the readers of Retrovirology find it equally thought-provoking. i Carol Carter: Biography Carol Carter, PhD, is a professor in the Department of Microbiology and Immunology at Stony Brook University Renaissance School of Medicine (Fig. 1 Carter bio photo Sebla B. Kutluay: Biography Sebla B. Kutluay, PhD, is an assistant professor in the Department of Molecular Microbiology at Washington University School of Medicine (Fig. Dr. Kutluay conducted her postdoctoral studies in the lab of Dr. Paul Bieniasz at Rockefeller University, where she made seminal discoveries in HIV-1 particle assembly, maturation, selective genome packaging and virus-host interactions. I Retrovirology is pleased to inaugurate a regular segment called "Interview with a Retrovirologist", in which two scientists discuss their careers, with the goal of highlighting leaders and rising stars, celebrating diversity and inspiring the next generation of scientists. [Extracted from the article]

Dr. Kutluay conducted her postdoctoral studies in the lab of Dr. Paul Bieniasz at Rockefeller University, where she made seminal discoveries in HIV-1 particle assembly, maturation, selective genome packaging and virus-host interactions. 1 Carter bio photo Sebla B. Kutluay: Biography Sebla B. Kutluay, PhD, is an assistant professor in the Department of Molecular Microbiology at Washington University School of Medicine (Fig. We learned a lot about both Carol and Sebla from this piece, and hope that the readers of Retrovirology find it equally thought-provoking. i Carol Carter: Biography Carol Carter, PhD, is a professor in the Department of Microbiology and Immunology at Stony Brook University Renaissance School of Medicine (Fig. I Retrovirology is pleased to inaugurate a regular segment called "Interview with a Retrovirologist", in which two scientists discuss their careers, with the goal of highlighting leaders and rising stars, celebrating diversity and inspiring the next generation of scientists. [Extracted from the article]

The aim of this article is to provide primary teachers with effective ways of engaging in drama processes to affirm diversity and to promote inclusive education in regard to cultural and linguistic diversity. It is informed by, and a response to, the ‘cultural and linguistic’ section within the recently revised version of theDrama Australia Equity and Diversity Guidelinesto which both authors contributed. It examines some ways in which recommendations and advice provided within the document can be enacted in primary schools through the use of drama education methods, in particular dramatic storytelling and process drama. Through their work the authors have found that drama techniques and processes can be highly effective in developing understandings of cultural and linguistic diversity within primary schools. By way of illustration this paper also focuses on the findings of a recent PhD project conducted by one of the authors, which focused on the education of pre-service primary teachers in the use of drama techniques to explore cultural identities and linguistic diversity. Specifically, it examined the roles culturally specific oral arts forms, including participatory storytelling (in this instance stories from South African cultures) can play in supporting drama pedagogy and intercultural understanding of pre-service primary teacher candidates and how they can transfer this knowledge to their teaching. [ABSTRACT FROM PUBLISHER]

Despite many changes made in 2010 and 2011 to Medicare's payment system for short-term stays in skilled nursing facilities, a flawed payment structure continues to underpay facilities for certain types of patients and overpay for others. The flaws in the payment structure create incentives to selectively admit or refuse patients based on the type and complexity of their conditions, while payments that vary with level of use encourage providers to furnish therapy services, such as rehabilitation care, that some patients might not need. We propose an alternative payment design and demonstrate that it would dampen such incentives by making payments that are more closely matched to costs and based on characteristics of the patients treated. We propose replacing the existing therapy component of payment with one that varies payments according to the expected care needs of the patient and adding a separate payment component that covers drugs and other nontherapy ancillary services, such as support for patients on ventilators. We also propose adding an outlier policy to provide additional reimbursement for patients requiring exceptionally high cost care. [ABSTRACT FROM AUTHOR]

More than a decade has elapsed since the link between the endosomal sorting complex required for transport (ESCRT) machinery and HIV-1 protein trafficking and budding was first identified. L domains in HIV-1 Gag mediate recruitment of ESCRT which function in bud abscission releasing the viral particle from the host cell. Beyond virus budding, the ESCRT machinery is also involved in the endocytic pathway, cytokinesis, and autophagy. In the past few years, the number of non-ESCRT host proteins shown to be required in the assembly process has also grown. In this paper, we highlight the role of recently identified cellular factors that link ESCRT machinery to calcium signaling machinery and we suggest that this liaison contributes to setting the stage for productive ESCRT recruitment and mediation of abscission. Parallel paradigms for non-ESCRT roles in virus budding and cytokinesis will be discussed. [ABSTRACT FROM AUTHOR]

In their recent publication, Rossman et al. [1] describe how the inherent budding capability of its M2 protein allows influenza A virus to bypass recruitment of the cellular ESCRT machinery enlisted by several other enveloped RNA and DNA viruses, including HIV, Ebola, rabies, herpes simplex type 1 and hepatitis B. Studies from the same laboratory [2] and other laboratories [3-6] indicate that budding of plasmid-derived virus-like particles can be mediated by the influenza virus hemagglutinin and neuraminidase proteins in the absence of M2. These events are also independent of canonical ESCRT components [2,7]. Understanding how intrinsic properties of these influenza virus proteins permit ESCRT-independent budding expands our understanding of the budding process itself. [ABSTRACT FROM AUTHOR]

HIV infection of macrophages is a critically important component of viral pathogenesis and progression to ATDS. Although the virus follows the same life cycle in macrophages and T lymphocytes, several aspects of the virus-host relationship are unique to macrophage infection. Examples of these are the long-term persistence of productive infection, sustained by the absence of cell death, and the ability of progeny virus to bud into and accumulate in endocytic compartments designated multivesicular bodies (MVBs). Recently, the hypothesis that viral exploitation of the macrophage endocytic machinery is responsible for perpetuating the chronic state of infection unique to this cell type has been challenged in several independent studies employing a variety of experimental strategies. This review examines the evidence supporting and refuting the canonical hypothesis and highlights recently identified cellular factors that may contribute to the unique aspects of the HIV-macrophage interaction. [ABSTRACT FROM AUTHOR]

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed. [Copyright &y& Elsevier]

Examines the mutagenesis and kinetics of plant cysteine proteinase using unusual arrangement of acidic amino acids. Details on the experiment on Ananain; Comparison of the kinetics of wild-type ananain with carboxyl residues E50A and E35A mutants; Role of acidic residues in the enhancement of catalytic competence.

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101–Ub–Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding. [ABSTRACT FROM AUTHOR]

The tissue-specific and developmental expression of histone 2A was studied in onion (Allium cepa "Robusta"), using northern blots. Histone 2A expression was enriched in basal tissues, particularly in the inner, meristematically active parts of bulbs. The expression was assessed during a time course of bulb development, dormancy onset and post-harvest sprouting in field-grown material. During bulb development historic 2A expression in the inner bulb declined rapidly during bulb ripening, reaching a minimum with the onset of dormancy. During post-harvest storage, expression increased slowly, reaching a peak in the spring, coinciding with the first observed sprout emergence. It was concluded that in onion, as in other plant systems, histone 2A expression is linked to cell division and dormancy level, the peak in expression during post-harvest storage indicating the time of dormancy breakage. In cultivars where post-harvest sprouting occurred much earlier or much later than in "Robusta," this expression peak occurred at about the same time of year, regardless of sprouting time. It was concluded that differences in storage longevity between cultivars were not due to differing times of dormancy breakage. Factors controlling the rate of sprout emergence post-dormancy are likely to be major determinants of storage capacity. [ABSTRACT FROM AUTHOR]

Reports on the cloning of the immediate upstream kinase mitogen-activated protein kinase (MEK) from mouse pituitary cDNA. Activation by upstream kinases; Phosphotransferase activity toward MAPK; Difference in autophosphorylation; Ability to serve as a substrate for MAPK; Differences in potential regulatory mechanisms of MEK1 and MEK2/2b.

Reports of declining memory are common with increasing age. Sometimes these are corroborated by poor memory test performance, but often they appear to be more closely related to depressive symptomatology. As pharmacological treatments emerge for improving cognition in the elderly, understanding the aetiology of memory complaints will become increasingly important. This article compares memory performance, reports of memory loss and depressive symptoms in attenders at a GP-referral and a self-referral memory clinic, with age- and sex-matched community controls. The GP-referred patients were older, had lower MMSE scores and had levels of memory complaint and depression between the control and self-referred subjects. The self-referrers had cognitive test performance similar to community controls but complained more of memory loss, were more depressed and more frequently reported a past history, of treated depression. Self-presentation of memory complaint appears to be more closely related to affective and possibly personality factors than memory test performance. [ABSTRACT FROM AUTHOR]

Discusses the results of a project undertaken in the United States to investigate the attitudes of students of elementary teacher education classes, towards inner-city schools. Objectives of the project; Methods used and results of the project.

Determines the nucleotide sequence of a linear plasmid of Borrelia burgdorferi. Similarities to those of circular plasmids of other prokaryotes; DNA cloning, sequencing and analysis; Nucleotide composition; Possible origin of linear plasmids of Borrelia.

Examines the regulation of mitogen-activated protein kinases (MAPKs) from sequential activation of a series of protein kinases. MAP kinases (MEKs); MEK kinase (MEKK); MEKK protein expressed in COS-1 cells to define its function in regulating the signaling system that includes MAPK; More.

Recent studies implicate the vacuolar protein-sorting pathway in the transport of the retroviral structural precursor (Gag) protein to its budding site on the plasma membrane of infected cells. This exploitation of the cellular endocytic trafficking machinery to release viral particles could lead to the identification of virus-specific modulators and provide opportunities to design new targeted anti-viral agents. [ABSTRACT FROM AUTHOR]

Here, an in vitro characterization of a family of prazole derivatives that covalently bind to the C73 site on Tsg101 and assay their ability to inhibit viral particle production is presented. Structurally, increased steric bulk on the 4‐pyridyl of the prazole expands the prazole site on the UEV domain toward the β‐hairpin in the Ub‐binding site and is coupled to increased inhibition of virus‐like particle production in HIV‐1. Increased bulk also increased toxicity, which is alleviated by increasing flexibility. Further, the formation of a novel secondary Tsg101 adduct for several of the tested compounds and the commercial drug lansoprazole. The secondary adduct involved the loss of the 4‐pyridyl substituent to form an irreversible species, with implications for increasing the half‐life of the active species or its specificity toward Tsg101 UEV. It is also determined that sulfide derivatives display effective viral inhibition, presumably through cellular sulfoxidation, allowing for delayed conversion within the cellular environment, and identify SARS‐COV‐2 as a target of prazole inhibition. These results open multiple avenues for the design of prazole derivatives for antiviral applications. [ABSTRACT FROM AUTHOR]

The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cellexpressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

A retroviral capsid (CA) protein consists of two helical domains, CAN and CAC, which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a β-hairpin motif upon processing from its precursor polyprotein Gag. The β-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the β-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the β-hairpin structure demonstrated that folding of the β-hairpin specifically extended the N terminus of helix α1 from Tyr20 to Pro17. This coil to helix transition involves the conserved sequence of Thr16-Pro17-Arg18 (Ser16-Pro17-Arg18 in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CAN oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg18, helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the β-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CAN oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CAN domain oligomerization, independent to the faster EIAV-CACdomain dimerization. [ABSTRACT FROM AUTHOR]

Abstract: Human immunodeficiency virus type 1 (HIV-1) release efficiency is directed by late (L) domain motifs in the viral structural precursor polyprotein Gag, which serve as links to the ESCRT (endosomal sorting complex required for transport) machinery. Linkage is normally through binding of Tsg101, an ESCRT-1 component, to the P7TAP motif in the p6 region of Gag. In its absence, budding is directed by binding of Alix, an ESCRT adaptor protein, to the LY36PXnL motif in Gag. We recently showed that budding requires activation of the inositol 1,4,5-triphosphate receptor (IP3R), a protein that “gates” Ca2+ release from intracellular stores, triggers Ca2+ cell influx and thereby functions as a major regulator of Ca2+ signaling. In the present study, we determined whether the L domain links Gag to Ca2+ signaling machinery. Depletion of IP3R and inactivation of phospholipase C (PLC) inhibited budding whether or not Tsg101 was bound to Gag. PLC hydrolysis of phosphatidylinositol-(4,5)-bisphosphate generates inositol (1,4,5)-triphosphate, the ligand that activates IP3R. However, with Tsg101 bound, Gag release was independent of Gq-mediated activation of PLC, and budding was readily enhanced by pharmacological stimulation of PLC. Moreover, IP3R was redistributed to the cell periphery and cytosolic Ca2+ was elevated, events indicative of induction of Ca2+ signaling. The results suggest that L domain function, ESCRT machinery and Ca2+ signaling are linked events in Gag release. [Copyright &y& Elsevier]

Abstract: We reported recently that activation of the inositol 1,4,5-triphosphate receptor (IP3R) is required for efficient HIV-1 Gag trafficking and viral particle release. IP3R activation requires phospholipase C (PLC)-catalyzed hydrolysis of PI(4,5)P2 to IP3 and diacylglycerol. We show that Sprouty2 (Spry2), which binds PI(4,5)P2 and PLCγ, interfered with PI(4,5)P2 in a manner similar to that of U73122, an inhibitor of PI(4,5)P2 hydrolysis, suggesting that Spry2 negatively regulates IP3R by preventing formation of its activating ligand, IP3. Mutation to Asp of R252, a crucial determinant of PI(4,5)P2 binding in the C-terminal domain of Spry2, prevented the interference, indicating that binding to the phospholipid is required. By contrast, deletion of the PLCγ binding region or mutation of a critical Tyr residue in the region did not prevent the interference but Spry2-PI(4,5)P2 colocalization was not detected, suggesting that PLC binding is required for their stable association. Like U73122, Spry2 over-expression inhibited wild type Gag release as virus-like particles. Disrupting either binding determinant relieved the inhibition. IP3R-mediated Ca2+signaling, in turn, was found to influence Spry2 subcellular distribution and ERK, a Spry2 regulator. Our findings suggest that Spry2 influences IP3R function through control of PI(4,5)P2 and IP3R influences Spry2 function by controlling its distribution and ERK activation. [Copyright &y& Elsevier]

Members of the Nedd4 family of E3 ubiquitin ligases bind the L domain in avian sarcoma virus (ASV) Gag and facilitate viral particle release. Translational fusion of ASV Gag with an L domain deletion (Δp2b) to proteins that comprise ESCRT-I, -II, and -III (the endocytic sorting complexes required for transport) rescued both Gag ubiquitination and particle release from cells. The ESCRT-I factors Vps37C or Tsg101 were more effective in rescue of Gag/Δp2b budding than the ESCRT-II factor Eap20 or the ESCRT-III component CHMP6. Thus ESCRT components can substitute for Nedd4 family members in ASV Gag release. Unlike wild type, ASV Gag/Δp2b -ESCRT chimeras failed to co-immunoprecipitate with co-expressed hemagglutinin- tagged Nedd4, indicating that Nedd4 was not stably associated with these Gag fusions. Release of the Gag-ESCRT-I or -II fusions was inhibited by a dominant negative mutant of Vps4 ATPase similar to wild type ASV Gag. In contrast to ASV Gag, HIV-1 Gag containing an L domain inactivating mutation (P7L) was efficiently rescued by fusion to a component of ESCRT-III (Chmp6) but not ESCRT-II (Eap20). Depletion of the endogenous pool of Eap20 (ESCRT-II) had little effect on HIV-1 Gag release but blocked ASV Gag release. In contrast, depletion of the endogenous pool of Vps37C (ESCRT-I) had little effect on ASV but blocked HIV-1 Gag release. Furthermore, an N-terminal fragment of Chmp6 inhibited both HIV-1 and ASV Gag release in a dominant negative manner. Taken together, these results indicate that ASV and HIV-1 Gag utilize different combinations of ESCRT proteins to facilitate the budding process, although they share some common elements. [ABSTRACT FROM AUTHOR]

Immature HIV-1 virions have spherical cores which become conical due to cleavage of the capsid domain of Gag. Here, we have used an immature form of capsid and show by electron microscopy, atomic force microscopy and single angle light scattering that it aggregates to spherical cores resembling immature virions at high ionic strengths and at pH values above 6. Dynamic angle light scattering of the dissociated protein shows structural changes that promote oligomerization above pH 6. We then examined the role of the required host protein cyclophilin A on assembly. Cyclophilin A is incorporated into virions at a 1:10 cyclophilin A/capsid ratio. We find that although cyclophilin A does not affect the oligomerization rate or stability of immature capsid cores, it does bind strongly to immature capsid at physiological stoichiometry above pH 6. This association serves as an entry route of cyclophilin A into HIV-1 virions. [Copyright &y& Elsevier]

Assembly of the human immunodeficiency virus type 1 (HIV-1) first occurs on the plasma membrane of host cells where binding is driven by strong electrostatic interactions between the N-terminal matrix (MA) domain of the structural precursor polyprotein, Gag, and the membrane. MA is also myristylated, but the exact role this modification plays is not clear. In this study, we compared the protein oligomerization and membrane binding properties of Myr[sup +] and Myr[sup -] Gag[sup MA] expressed in COS-1 cells. Sedimentation studies in solution showed that both the myristylated Gag precursor and the mature MA product were detected in larger complexes than their unmyristylated counterparts, and the myristylated MA protein bound liposomes with ∼3-fold greater affinity than unmyristylated MA. Aromatic residues near the N-terminal region of the MA protein were more accessible to chymotrypsin in the unmyristylated form and, consistent with this, an epitope in the N-terminal region was more exposed. Moreover, the cyclophilin binding site in the CA domain downstream of MA was more accessible in the unmyristylated Gag protein, while the Tsgl01 binding site in the C-terminal region was equally available in the unmyristylated and myristylated Gag proteins. Taken together, our results suggest that myristylation promotes assembly by inducing conformational changes and facilitating MA multimerization. This observation offers a novel role for myristylation. [ABSTRACT FROM AUTHOR]

Discusses the evolution of the functional diversity in cupin proteins. Characteristics of cupins; Details on the active site of cupins; Phylogeny of cupins.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus responsible for several diseases, including cancers of lymphoid and epithelial cells. EBV cancers typically exhibit viral latency; however, the production and release of EBV through its lytic phase are essential for cancer development. Antiviral agents that specifically target EBV production do not currently exist. Previously, we reported that the proton pump inhibitor tenatoprazole, which blocks the interaction of ubiquitin with the ESCRT-1 factor Tsg101, inhibits production of several enveloped viruses, including EBV. Here, we show that three structurally distinct prazoles impair mature particle formation postreactivation and identify the impact on stages of replication. The prazoles did not impair expression of lytic genes representative of the different kinetic classes but interfered with capsid maturation in the nucleus as well as virion transport from the nucleus. Replacement of endogenous Tsg101 with a mutant Tsg101 refractory to prazole-mediated inhibition rescued EBV release. These findings directly implicate Tsg101 in EBV nuclear egress and identify prazoles as potential therapeutic candidates for conditions that rely on EBV replication, such as chronic active EBV infection and posttransplant lymphoproliferative disorders. IMPORTANCE Production of virions is necessary for the ubiquitous Epstein-Barr virus (EBV) to persist in humans and can set the stage for development of EBV cancers in at-risk individuals. In our attempts to identify inhibitors of the EBV lytic phase, we previously found that a prazole proton pump inhibitor, known to block the interaction of ubiquitin with the ESCRT-1 factor Tsg101, blocks production of EBV. We now find that three structurally distinct prazoles impair maturation of EBV capsids and virion transport from the nucleus and, by interfering with Tsg101, prevent EBV release from lytically active cells. Our findings not only implicate Tsg101 in EBV production but also identify widely used prazoles as candidates to prevent development of posttransplant EBV lymphomas. [ABSTRACT FROM AUTHOR]

The Gag protein of avian sarcoma virus (ASV) lacks anN-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MAdomain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipaseC (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to thePMand budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to thePMwhen substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding. [ABSTRACT FROM AUTHOR]

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA87) to lipids and liposomes. We report that MA87 binds to the polar head of phosphoinositides such as PI(4,5)P2. We found that MA87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA87-IP binding is governed by charge-charge interactions. Using a sensitive NMRbased liposome-binding assay, we show that binding ofMA87 to liposomes is enhanced by incorporation of PI(4,5)P2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding ofMA87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly. [ABSTRACT FROM AUTHOR]

HIV-1 protease autoprocessing is responsible for liberation of free mature protease (PR) from the Gag-Pol polyprotein precursor. A cell-based model system was previously developed to examine the autoprocessing mechanism of fusion precursors carrying the p6*-PR miniprecursor sandwiched between various proteins or epitopes. We here report that precursor autoprocessing is context-dependent as its activity and outcomes can be modulated by sequences upstream of p6*-PR. This was exemplified by the 26aa maltose binding protein (MBP) signal peptide (SigP) when placed at the N-terminus of a fusion precursor. The mature PRs released from SigP-carrying precursors are resistant to self-degradation whereas those released from SigP-lacking fusion precursors are prone to self-degradation. A H69D mutation in PR abolished autoprocessing of SigP-containing fusion precursors whereas it only partially suppressed autoprocessing of fusion precursors lacking SigP. An autoprocessing deficient GFP fusion precursor with SigP exhibited a subcellular distribution pattern distinct from the one without it in transfected HeLa cells. Furthermore, a SigP fusion precursor carrying a substitution at the P1 position released the mature PR and PR-containing fragments that were different from those released from the precursor carrying the same mutation but lacking SigP. We also examined autoprocessing outcomes in viral particles produced by a NL4-3 derived proviral construct and demonstrated the existence of several PR-containing fragments along with the mature PR. Some of these resembled the SigP precursor autoprocessing outcomes. This finding of context-dependent modulation reveals the complexity of precursor autoprocessing regulation that most likely accompanies sequence variation imposed by the evolution of the upstream Gag moiety. [ABSTRACT FROM AUTHOR]

Background: The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. Results: Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. Conclusions: Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor. [ABSTRACT FROM AUTHOR]

Abstract: We previously demonstrated that ectodomain residue Asp286 in N2 neuraminidase (NA; Asp268 in N1 NA) present in budding-capable NA proteins contributes to productive NA plasma membrane transport partly by mediating escape from tetherin restriction [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480–2491]. Budding-incapable NA proteins contain a G at this position and either co-expression of human immunodeficiency virus type 1 vpu or siRNA-mediated depletion of tetherin rescued budding capabilities in these proteins [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480–2491]. Furthermore, replacement of D286 with G in budding-capable NA proteins caused loss of function, preventing release of NA virus-like particles (VLPs). Here, we show that mutation of this residue specifically modulates the ability of NA to escape tetherin restriction at the plasma membrane and results in virus attenuation in vivo. Based on immunogold electron microscopy and co-immunoprecipitation assays, both NAD286-containing and NAD286G-containing proteins associated with tetherin in the endoplasmic reticulum (ER). However, the NAD286G loss-of-function mutant also associated with the host factor outside the ER and in plasma-membrane-localized VLPs as visualized using immunogold electron microscopy. We conclude that the presence of aspartate at residue 286 liberates NA from tetherin-dependent restriction upon exit from the ER compartment thus preventing restriction at the plasma membrane. Underscoring the importance of these observations, knockdown of tetherin resulted in a 1–1.5 log increase in influenza virus growth. Additionally, the loss-of-function mutation conferred attenuation in a mouse model of influenza infection as evidenced by a 5-fold increase in LD50 and increases in either percent survival or time to death dependent on the administered dose in vivo. [Copyright &y& Elsevier]

Background HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and -2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants. Results Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and -2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage. Conclusions The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways. [ABSTRACT FROM AUTHOR]

We have determined that, in addition to its receptor-destroying activity, the influenza virus neuraminidase is capable of efficiently forming virus-like particles (VLPs) when expressed individually from plasmid DNA. This observation applies to both human subtypes of neuraminidase, N1 and N2. However, it is not found with every strain of influenza virus. Through gain-of-function and loss-of-function analyses, a critical determinant within the neuraminidase ectodomain was identified that contributes to VLP formation but is not sufficient to accomplish release of plasmid-derived VLPs. This sequence lies on the plasma membrane-proximal side of the neuraminidase globular head. Most importantly, we demonstrate that the antiviral restriction factor tetherin plays a role in determining the strain-specific limitations of release competency. If tetherin is counteracted by small interfering RNA knockdown or expression of the HIV anti-tetherin factor vpu, budding and release capability is bestowed upon an otherwise budding-deficient neuraminidase. These data suggest that budding-competent neuraminidase proteins possess an as-yet-unidentified means of counteracting the antiviral restriction factor tetherin and identify a novel way in which the influenza virus neuraminidase can contribute to virus release. [ABSTRACT FROM AUTHOR]

The ESCRT-I protein Tsg101 plays a critical role in viral budding and endocytic sorting. Although Tsg101 is known to recognize monoubiquitin (Ub 1), here we show that it can also bind several diubiquitins (K48-Ub 2 , N-Ub 2 , and K63-Ub 2), with a preference for K63-linked Ub 2. The NMR structure of the Tsg101:K63-Ub 2 complex showed that while the Ub 1 -binding site accommodates the distal domain of Ub 2 , the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site results in distinct phenotypes regarding the recruitment of Tsg101 partners. Mutation in the vestigial active site abrogates interaction between Tsg101 and the HIV-1 protein Gag but not Hrs, a cellular protein. Mutation at the N-terminal helix alters Gag but not Hrs-Tsg101 localization. Given the broad involvement of Tsg101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo. [Display omitted] • The Tsg101:K63-linked di-ubiquitin structure is solved by NMR • Pseudocontact shifts are used to dock and refine the structure of the complex • Lanthanide incorporation into specific ubiquitin (Ub) could distinguish between two Ub domains • Two different conformations of the diubiquitin are required to satisfy the NMR data Strickland et al. determine the structure of the K63 linked diubiquitin bound to Tsg101 UEV domain. The structure reveals the distal ubiquitin binds the monoubiquitin site on UEV, whereas the proximal ubiquitin can occupy two distinct sites. The two conformations of the diubiquitin on UEV have different functional consequences. [ABSTRACT FROM AUTHOR]

Abstract: The Late (L) domain of the avian sarcoma virus (ASV) Gag protein binds Nedd4 ubiquitin ligase E3 family members and is the determinant of efficient virus release in avian and mammalian cells. We previously demonstrated that Nedd4 and Tsg101 constitutively interact raising the possibility that Nedd4 links ASV Gag to the ESCRT machinery. We now demonstrate that covalently linking Tsg101 to ASV Gag lacking the Nedd4 binding site (Δp2b-Tsg101) ablates the requirement for Nedd4, but the rescue of budding occurs by use of a different budding mechanism than that used by wild type ASV Gag. The evidence that Tsg101 and Nedd4 direct release by different pathways is: (i) Release of the virus-like particles (VLPs) assembled from Gag in DF-1, an avian cell line, was resistant to dominant-negative interference by a Tsg101 mutant previously shown to inhibit release of both HIV and Mo-MLV. (ii) Release of VLPs from DF-1 cells was resistant to siRNA-mediated depletion of the endogenous pool of Tsg101 in these cells. (iii) VLPs assembled from wild-type ASV Gag exhibited highly efficient release from endosome-like membrane domains enriched in the tetraspanin protein CD63 or a fluorescent analogue of the phospholipid phosphatidylethanolamine. However, the VLPs assembled from the L domain mutant Δp2b or a chimeric Δp2b-Tsg101 Gag lacked these domain markers even though the chimeric Gag was released efficiently compared to the Δp2b mutant. These results suggest that Tsg101 and Nedd4 facilitate Gag release through functionally exchangeable but independent routes and that Tsg101 can replace Nedd4 function in facilitating budding but not directing through the same membranes. [Copyright &y& Elsevier]

Budding of retroviruses requires the structural precursor polyprotein, Gag, to target the plasma membrane through its N-terminal matrix (MA) domain. For HIV-1, the interaction between membrane signaling molecule phosphatidylinositol 4,5-diphosphate (PIP2) and MA induces the exposure of myristate and promotes membrane binding. Here we studied oligomerization of the naturally unmyristylated equine infectious anemia virus (EIAV) MA and its interaction with PIP2-C4 primarily using solution NMR spectroscopy. The measured 1H–15N residual dipolar coupling agrees with the atomic coordinates from the EIAV MA crystal structure. The analytical ultracentrifugation results show a dominant population of monomeric EIAV MA at a concentration of 63 μM and 20 °C, along with a small trimer and a broad distribution of other oligomers. The monomer–trimer equilibrium model and the quaternary packing of the trimer were further established by the concentration-dependent 15N spin relaxation rates and chemical shifts. Binding of MA to PIP2-C4 was detected by chemical shift mapping (CSM) with an apparent Kd of 182 ± 56 μM, a value similar to that reported for HIV-1 MA. The PIP2 binding site includes the Loop region between Helix2 and Helix3 in the EIAV MA. CSM and spin relaxation dispersion reveal a coupling of conformational change and submillisecond dynamics, respectively, between the Loop and trimeric Interface Residues due to PIP2 binding. We infer that PIP2 participates in the initial trimer formation of EIAV MA, but more importantly, the concentration effect is dominant in shifting the equilibrium toward trimer, in line with the entropic switch mechanism proposed for myristylated HIV-1 MA. [ABSTRACT FROM AUTHOR]

The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interfere with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co-expression with mutated forms of Vps4, Nedd4 and Tsg101. A dominant-negative mutant of Vps4 A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV-1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 did not prevent interaction; however, three-dimensional spatial imaging suggested that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 was not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101–Nedd4 interaction. [ABSTRACT FROM AUTHOR]

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal β-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the β-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus 13-hairpin by disrupting the salt bridge between Pro¹ and Asp54 that stabilizes the β-hairpin. NMR 15N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the β-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the β-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation. [ABSTRACT FROM AUTHOR]

Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA 98:11199–11204, 2001). We have now identified the complete 3′ end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag. [ABSTRACT FROM AUTHOR]